Hypoxia in animals activates a broad range of homeostatic responses via induction of a transcriptional complex termed hypoxia inducible factor (HIF). HIF is a heterodimer of α- and β-subunits, with regulation by dioxygen availability being mediated by post-translational modification of the α-subunits. In mammalian cells, at least two HIF-α subunit isoforms (HIF-1α and HIF-2α) are regulated by dioxygen levels. Each HIF-α protein contains an internal oxygen dependent degradation domain (ODDD) possessing targeting motifs for proteolytic regulation and a C-terminal transactivation domain (CAD) independently regulated by dioxygen, irrespective of changes in protein abundance, through interaction with the CH1 domain of the co-activator p300.
The oxygen dependent degradation of HIF-α by proteolysis is regulated by the hydroxylation of specific prolyl residues (Pro-402 and Pro-564 in human HIF-1α) that mediate recognition of HIF-α by the von Hippel-Lindau (VHL) ubiquitinylation complex, and consequent proteasomal destruction. Combined structural analysis and genetic approaches led to the identification of three isoforms of human HIF prolyl hydroxylase (PHD1-3, prolyl hydroxylase domain) together with homologues in a range of organisms. In vitro analyses, together with sequence and mutational analyses identified these as belonging to a sub-family of the Fe(II) and 2-oxoglutarate (2-OG) dependent oxygenases. Limiting oxygen availability in hypoxia, or direct inhibition of the PHD enzymes by cobaltous ions and iron chelators, allows HIF-α to escape hydroxylation and recognition by pVHL, providing insights into the mechanism by which these stimuli suppress HIF-αdegradation and activate the transcriptional cascade.
Previous analyses of the HIF-α CAD have indicated that, as with proteolysis, the action of hypoxia is mimicked by cobaltous ions and iron chelators.
Mass spectrometric and mutational analyses of the HIF-α CAD demonstrate regulatory hydroxylation of a specific asparaginyl residue (Asn-803 in HIF-1α). In the presence of oxygen, hydroxylation at this site prevents interaction with the p300 CH1 domain, whereas in hypoxia suppression of the modification allows interaction with p300 and transcriptional activation. Consistent with this model, NMR studies of the human HIF-1α CAD complexed to CH1 indicate that the unmodified Asn-803 is buried at the interface between the proteins.